Bioactive small molecules, proteins and peptides are routinely conjugated with water soluble polymers to increase their water solubility, circulation half life and decrease the antigenicity. Amongst many water soluble polymers poly(ethylene glycol) (PEG) is particularly useful in modification of pharmaceutically active compounds, proteins and peptides. “Pegylated” conjugates often display improved pharmacokinetic properties than native bioactive agents. A rich variety of pegylation reagents is commercially available for chemical modification of bioactive agents. Linear, branched, star shaped, and multi-arm pegylation reagents all have been used [Harris J M, Chess R B. Effect of pegylation on pharmaceuticals. Nature 2003; 2: 214-221; Vicent M J, Duncan R. Polymer conjugates: Nanosized medicines for treating cancer. Trends in Biotechnol. 2006; 24: 39-47; Duncan R. The dawning of polymer therapeutics. Nature Rev. Drug Discovery 2003; 2: 347-360; Sheffield W P. Modification of clearance of therapeutic and potentially therapeutic proteins. Current Drug Targets—Cardiovas. & Hemat. Dis. 2001; 1: 1-22; Veronese F M, Pasut G. PEGylation, successful approach to drug delivery. Drug Deliv. Today 2005; 10: 1451-1458; Mehvar R. Modulation of pharmacokinetics and pharmacodynamics of proteins by polyethylene glycol conjugation. J. Pharm. Pharmceut. Sci. 2000; 3: 125-136; Kozlowski A, Harris J M. Improvements in protein PEGylation: pegylated intereferons for treatment of hepatitis C. J. Controlled Rel. 2001; 72: 217-224].
Branched pegylation reagents have proven to be particularly useful over linear pegylation reagents as these reagents provide either multiple sites for conjugation of bioactive molecules or multiple PEG chains conjugated per bioactive molecule. Also, once conjugated to a therapeutic, the branched polymers have provided longer circulation times than that provided by linear polymers. However, syntheses of multi-arm or branched pegylation reagents described in the prior art has involved use of tedious purification procedures and laborious chemistries of protection and deprotection of multiple functional groups that need to be modified with either PEG chains or bioactive molecules. Also despite such tedious chemistries, the reagents described in the prior art have had very few attachment points for polymers and bioactive agents.
For example, U.S. Pat. No. 7,291,713 describes preparation of three chain-branched pegylation reagents by modifying pentaerythritol which contains four primary hydroxyl groups. According to this invention, an excess of PEG is conjugated to pentaerythritol and a fraction in which PEG is conjugated to only three out of four available hydroxyl groups is isolated by column chromatography. Then the remaining hydroxyl group is modified with a linker to prepare the final, branched pegylation reagent.
Another U.S. Pat. No. 7,316,811 describes multiarm PEG-polypeptide block copolymers for conjugating multiple drug molecules to polymer carriers. According to this invention, a block copolymer between 8-arm PEG and poly(benzyl aspartate) or poly(benzyl glutamate) is first prepared. Benzyl groups on block copolymer are deprotected and the drug is conjugated with free carboxyl groups of poly(aspartic acid) or poly(glutamic acid) segment in the block copolymer.
U.S. Pat. No. 6,566,506 describes branched PEG reagent synthesized by conjugating two primary amino groups in 1,3-diamino-2-propanol selectively with PEG chains and the secondary hydroxyl group with para-nitrophenyl chloroformate, without applying protection-deprotection chemistries. The branched PEG reagent was synthesized by exploiting reactivity differences between amino and hydroxyl groups in conjugation reactions. However, the limitations in using commonly available amino alcohols as starting molecules for synthesis of branched PEG reagents is that these amino alcohols contain very few attachment sites for polymers and bioactive agents.
Yet another U.S. Pat. No. 7,026,440 describes preparation of multiarm pegylation reagents using hydrocarbon moieties with multiple functional groups attached to PEG chains and a linker moiety capable of conjugating at a single point. Glycerol is the preferred hydrocarbon used here. Secondary hydroxyl groups in glycerol were first protected and PEG chains were grown from the two free primary hydroxyls via ethylene oxide polymerization. The terminal hydroxyls on grown PEG chains were then protected, the secondary hydroxyl was deprotected, and a linker was finally attached. Briefly, this chemistry provides reagents containing multiple PEG chains and a single attachment site within a single molecule. Reagents that provide multiple PEG chains and multiple sites for attachment of bioactive agents are not achievable from this art.
In summary, prior art provides limited structural and conformational variety of central aliphatic core in branched pegylation reagents and few sites for attachment of bioactive agents. The main reason for this shortcoming is lack of multifunctional, core forming molecules to which polymers and biomolecules can be conjugated selectively and preferably with simpler chemistries. It is therefore the object of this invention to provide a simple method for synthesis of multifunctional, activated building blocks. It is also the object of this invention to provide such multifunctional, activated building blocks and their conjugates with polymers and bioactive agents.